The invention relates to the use of yacM and yqeJ, which are essential bacterial genes, in identifying antibacterial agents.
Bacterial infections may be cutaneous, subcutaneous, or systemic. Opportunistic bacterial infections can be life threatening, especially in patients afflicted with AIDS or other diseases that compromise the immune system. Most bacteria that are pathogenic to humans are gram-positive bacteria. The bacterium Streptococcus pneumoniae, for example, typically infects the respiratory tract and can cause lobar pneumonia, as well as meningitis, sinusitis, and other infections.
The invention is based on the discovery that the yacM and yqeJ genes of the gram positive bacterium Streptococcus pneumoniae, termed xe2x80x9cS-yacMxe2x80x9d and xe2x80x9cS-yqeJ,xe2x80x9d are essential for survival. Thus, the essential polypeptides that these genes encode are useful targets for identifying compounds that are inhibitors of the bacteria in which the polypeptides are expressed. Such inhibitors can inhibit bacterial growth by inhibiting the activity of an essential protein, or by inhibiting transcription of an essential gene or translation of the mRNA transcribed from the essential gene. The amino acid and nucleic acid sequences of the Streptococcus yacM and yqeJ polypeptides and genes are set forth in FIGS. 1 and 2, as summarized in Table 1.
Identification of these essential genes allows homologs of the essential genes to be found in other strains within the species, and it allows orthologs of the essential genes to be found in other organisms (e.g., Bacillus ssp. and E. coli). The terms xe2x80x9cyacMxe2x80x9d and xe2x80x9cyqeJxe2x80x9d refer to the S-yacM and S-yqeJ genes and polypeptides, as well as their homologs and orthologs, collectively. While xe2x80x9chomologsxe2x80x9d are structurally similar genes contained within a species, xe2x80x9corthologsxe2x80x9d are functionally equivalent genes from other species (within or outside of a given genus). The yacM and yqeJ genes and polypeptides can be used in methods for identifying similar genes in pathogenic and non-pathogenic microorganisms. In particular, S-yacM and S-yqeJ genes can be used to identify orthologs of yacM and yqeJ genes in other species (e.g., other gram positive bacteria, and other bacteria generally). Examples of orthologs of these Streptococcus genes are summarized in Table 2. As shown in Table 2, the Streptococcus yacM gene has an ortholog in B. subtilis, termed xe2x80x9cB-yacM,xe2x80x9d and an ortholog in E. coli, termed xe2x80x9cygbP.xe2x80x9d The Streptococcus yqeJ gene also has an ortholog in B. subtilis, termed xe2x80x9cB-yqeJ,xe2x80x9d and an ortholog in E. coli, termed xe2x80x9cybeN.xe2x80x9d Having identified such orthologous genes as essential, these orthologous genes and the polypeptides encoded by these orthologs can be used to identify compounds that inhibit the growth of the host organism (e.g., compounds that inhibit the activity of an essential protein, or inhibit transcription of an essential gene).
The yacM and yqeJ polypeptides and genes described herein include the polypeptides and genes set forth in FIGS. 1 and 2 herein, as well as isozymes, allelic variants, and conservative variants of the sequences set forth in FIGS. 1 and 2. For example, the invention includes a gene that encodes a yacM or yqeJ polypeptide but which gene includes one or more point mutations, deletions, or promoter variants, provided that the resulting essential polypeptide retains a biological function of a yacM or yqeJ polypeptide as determined, for example, in a conventional complementation assay. Also encompassed by the terms yacM gene and yqeJ gene are degenerate variants of the nucleic acid sequences set forth in FIGS. 1-6. Degenerate variants of a nucleic acid sequence exist because of the degeneracy of the amino acid code; thus, those sequences that vary from the sequences shown in FIGS. 1-6, but which nonetheless encode a yacM or yqeJ polypeptide are degenerate variants.
Likewise, because of the similarity in the structures of amino acids, conservative variations (as described herein) can be made in the amino acid sequence of the yacM or yqeJ polypeptide while retaining the function of the polypeptide (e.g., as determined in a conventional complementation assay). Other yacM and yqeJ polypeptides and genes identified in additional bacterial strains may be such conservative variations or degenerate variants of the particular yacM or yqeJ polypeptide and nucleic acid set forth in FIGS. 1-6 (SEQ ID NOs:1-18). The yacM genes and polypeptides share at least 80%, e.g., 90% or 95%, sequence identity with the sequences shown in FIGS. 1, 3, or 4. Regardless of the percent sequence identity between the yacM sequence and the sequence shown in FIGS. 1, 3 or 4, the yacM genes and polypeptides that can be used in the methods of the invention preferably are able to complement for the lack of yacM function (e.g., in a temperature-sensitive mutant) in a standard complementation assay.
Additional yacM genes that are identified and cloned from additional bacterial strains, and pathogenic, gram positive strains in particular, can be used to produce yacM polypeptides for use in the various methods described herein, e.g., for identifying antibacterial agents. Likewise, the term xe2x80x9cyqeJxe2x80x9d encompasses isozymes, variants, and conservative variations of the sequences depicted in FIGS. 2, 5, or 6.
In various embodiments, the essential polypeptide used in the assays described herein is derived from a non-pathogenic or pathogenic gram-positive bacterium. For example, the polypeptide can be derived from a Streptococcus strain, such as Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus endocarditis, Streptococcus faecium, Streptococcus sangus, Streptococcus viridans, and Streptococcus hemolyticus. Suitable orthologs of the essential genes can be derived from a wide spectrum of bacteria, such as E. coli and Bacillus subtilis. 
Because the genes described herein have been shown to be essential for survival, the essential genes and polypeptides encoded by these essential genes, as well as their homologs and orthologs, can be used to identify antibacterial agents. Such antibacterial agents can readily be identified with high throughput assays to detect inhibition of the metabolic pathway in which the essential polypeptide participates. This inhibition can be caused by small molecules interacting with (e.g., binding directly or indirectly to) the essential polypeptide or other essential polypeptides in that pathway.
In an exemplary assay, but not the only assay, a promoter that responds to depletion of the essential polypeptide by upregulation or downregulation is linked to a reporter gene (e.g., xcex2-galactosidase, gus, or GFP), as described herein. A bacterial strain containing this reporter gene construct is then exposed to test compounds. Compounds that inhibit the yacM or yqeJ polypeptide (or other polypeptides in the essential pathway in which the polypeptide participates) will cause a functional depletion of the yacM or yqeJ polypeptide and therefore lead to an upregulation or downregulation of expression of the reporter gene. Because the polypeptides described herein are essential for the survival of bacteria, compounds that inhibit the essential polypeptides in such an assay are candidate antibacterial compounds and can be further tested, if desired, in conventional susceptibility assays to determine if these compounds are antibacterial agents.
In other suitable methods, screening for antibacterial agents is accomplished by (i) identifying those compounds that interact with or bind to a yacM or yqeJ polypeptide and (ii) further testing such compounds for their ability to inhibit bacterial growth in vitro or in vivo.
The invention also provides methods of preparing an antibacterial agent. The methods include: screening multiple test compounds by the methods described herein; identifying candidate antibacterial compounds that interact with yacM or yqeJ; isolating one or more lead compounds from the candidate compounds; identifying a lead compound that inhibits bacterial growth; selecting a lead compound that inhibits bacterial growth; and formulating the selected lead compound as an antibacterial agent. A xe2x80x9clead compoundxe2x80x9d is a test compound that binds to yacM or yqeJ with a statistically significant binding affinity. For example, the binding can be sufficient to inhibit an essential function of the protein.
If desired, lead compounds can subsequently be derivatized using conventional medicinal chemistry methods, as described herein. Thus, the invention includes methods for preparing an antibacterial agent by: screening multiple test compounds by the methods described herein; identifying candidate compounds that interact with yacM or yqeJ; isolating one or more lead compounds from the candidate compounds; derivatizing the lead compound(s), thereby producing a derivative of the lead compound; identifying derivatives that inhibit bacterial growth; and formulating the derivative as an antibacterial agent (e.g., by admixture with a pharmaceutically acceptable carrier). Antibacterial agents prepared by such methods also are included within the invention and can be used in methods for treating a bacterial infection in an organism.
Typically, the test compound will be a small organic molecule. Alternatively, the test compound can be a test polypeptide (e.g., a polypeptide having a random or predetermined amino acid sequence; or a naturally-occurring or synthetic polypeptide) or a nucleic acid, such as a DNA or RNA molecule. The test compound can be a naturally-occurring compound or it can be synthetically produced, if desired. Synthetic libraries, chemical libraries, bodily fluids (e.g., urine, sweat, tears, blood, or CSF) and the like can be screened to identify compounds that bind to the yacM or yqeJ polypeptide. More generally, binding of a test compound to the polypeptide can be detected either in vitro or in vivo. If desired, the above-described methods for identifying compounds that modulate the expression of the polypeptides of the invention can be combined with measuring the levels of the yacM or yqeJ polypeptide expressed in the cells, e.g., by performing a Western blot analysis using antibodies that bind to a yacM or yqeJ polypeptide.
Regardless of the source of the test compound, the yacM and yqeJ polypeptides described herein can be used to identify compounds that inhibit (i) the activity of a yacM or yqeJ protein, (ii) transcription of a yacM or yqeJ gene, or (iii) translation of the mRNA transcribed from the yacM or yqeJ gene. These antibacterial agents can be used to inhibit a wide spectrum of pathogenic or non-pathogenic bacterial strains.
In other embodiments, the invention includes pharmaceutical formulations that include a pharmaceutically acceptable excipient and an antibacterial agent identified using the methods described herein. In particular, the invention includes pharmaceutical formulations that contain antibacterial agents that inhibit the growth of, or kill, pathogenic bacterial strains (e.g., pathogenic gram positive bacterial strains such as pathogenic Streptococcus strains). Such pharmaceutical formulations can be used in a method of treating a bacterial infection in an organism (e.g., a Streptococcus infection). Such a method entails administering to the organism a therapeutically effective amount of the pharmaceutical formulation, i.e., an amount sufficient to ameliorate signs and/or symptoms of the bacterial infection. In particular, such pharmaceutical formulations can be used to treat bacterial infections in mammals such as humans and domesticated mammals (e.g., cows, pigs, horses, dogs, and cats), and in plants. The efficacy of such antibacterial agents in humans can be reasonably predicted by using animal model systems well known to those of skill in the art (e.g., mouse and rabbit model systems of, for example, streptococcal pneumonia).
Purified or isolated antibodies that specifically bind to a yacM or yqeJ polypeptide can be used in the methods of the invention. An antibody xe2x80x9cspecifically bindsxe2x80x9d to a particular antigen, e.g., a yacM or yqeJ polypeptide, when it binds to that antigen, but does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample, that naturally includes a yacM or yqeJ polypeptide.
The invention offers several advantages. For example, the methods for identifying antibacterial agents can be configured for high throughput screening of numerous candidate antibacterial agents. Because the yacM and yqeJ genes disclosed herein are thought to be highly conserved, antibacterial drugs targeted to these genes or their gene products are expected to have a broad spectrum of antibacterial activity.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the case of a conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative and are not intended to limit the scope of the invention, which is defined by the claims.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.